Sains Malaysiana 48(8)(2019):
1671–1683
http://dx.doi.org/10.17576/jsm-2019-4808-13
The Effect of Stichopus chloronotusAqueous
Extract on Human Osteoarthritis Articular Chondrocytes in Three-Dimensional
Collagen Type I Hydrogel in vitro
(Kesan
Ekstrak Akues
Stichopus chloronotuspada Kondrosit Osteoartritis
Artikul Manusia
dalam Kolagen Tiga Dimensi Hidrogel Jenis I secara in vitro)
MOHD HEIKAL MOHD YUNUS1,4*, AHMAD NAZRUN SHUID2, MOHD FAUZI BUSRA4, CHUA KIEN HUI1, NORZANA ABDUL GHAFAR3 & RIZAL ABD RANI5
1Department of
Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala
Lumpur, Federal Territory, Malaysia
2Department of
Pharmacology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala
Lumpur, Federal Territory, Malaysia
3Department of Anatomy, Universiti Kebangsaan Malaysia
Medical Centre, Jalan Yaacob Latiff, Bandar Tun Razak, 56000 Cheras, Kuala
Lumpur, Federal Territory, Malaysia
4Tissue Engineering
Centre, Universiti Kebangsaan Malaysia Medical Center, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras Kuala Lumpur, Federal Territory, Malaysia
5Department of Orthopaedic, Universiti Kebangsaan Malaysia Medical Center, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras Kuala Lumpur,
Kuala Lumpur, Federal Territory, Malaysia
Diserahkan: 25 September 2018/Diterima: 6 Mei 2019
ABSTRACT
Autologous
chondrocyte-seeded scaffolds have proved to be one of the most promising
alternative therapies for articular cartilage defects. However, the
chondrocytes have specific nutritional requirements and risk of
dedifferentiation during in vitro expansion. Stichopus chloronotus aqueous
extract (SCAE) was investigated as a medium supplement for
three-dimensional (3D) collagen type I hydrogel scaffold seeded with
chondrocytes to determine whether SCAE is capable of maintaining
phenotype and sustaining extracellular matrix synthesis and deposition. Human
osteoarthritis articular chondrocytes were isolated from the knee joint
cartilage of patients underwent total knee replacement surgery. Human
osteoarthritis articular chondrocytes were encapsulated in collagen type I
hydrogel and cultured in basic medium with 0, 0.1 and 0.2% of SCAE.
The chondrocytes in 3D culture were evaluated by means cell morphology and
proliferation, quantitative phenotypic expression of collagen type I, II, aggrecan core protein and sox-9. H&E, toluidine blue
staining and sulfated glycosaminoglycan (sGAG)
production were analyzed after 7 days in culture. Chondrocytes cultured in 3D
with various SCAE concentration appeared with polygonal morphology
maintaining their chondrocytes characteristic. SCAE supplementation
promoted chondrocytes proliferation and the ability of the cells to express
gene encoding collagen type I decreased, whereas their ability to express
collagen type II, aggrecan core protein and sox9
increased as compared to control. The cartilaginous matrices were positively
stained toluidine blue concomitant with higher sGAG accumulation
in SCAE-supplemented culture medium. This study shown that SCAE may be beneficial for in vitro development of 3D
chondrocytes culture for use in cartilage tissue engineering therapies.
Keywords: Chondrocytes;
collagen Type I; osteoarthritis; Stichopus chloronotus;
three dimensional culture
ABSTRAK
Biobahan yang disemai kondrosit
secara autologus
telah terbukti menjadi salah satu
terapi alternatif
yang paling berkesan untuk
kecacatan rawan artikul. Walau bagaimanapun, kondrosit mempunyai keperluan nutrien tertentu dan berisiko untuk
mengalami nyahpembezaan semasa pengembanganin vitro. Ekstrak air Stichopus chloronotus (SCAE)
telah dikaji
sebagai tambahan bagi media untuk kondrosit yang disemai dalam hidrogel kolagen jenis I secara tiga dimensi
(3-D) untuk menentukan
sama ada SCAE mampu mengekalkan fenotip dan sintesis
serta pemendapan
matriks ekstrasel. Kondrosit osteoartritis manusia dipencilkan daripada rawan sendi lutut pesakit
yang menjalani pembedahan
penggantian lutut secara total. Kondrosit osteoartritis manusia disemai dalam hidrogel
kolagen jenis
I dan dikultur dalam
medium asas dengan
0, 0.1 dan 0.2% SCAE. Kondrosit
yang dikultur secara
3D dinilai daripada segi morfologi dan percambahan sel, ekspresi fenotip
kuantitatif bagi
kolagen jenis I, II, protein
teras agrekan dan
sox-9. Pewarnaan H&E dan
toluidin biru
serta penghasilan glikosaminoglikan tersulfat
(sGAG) dianalisis selepas dikultur selama 7 hari. Kondrosit yang dikultur secara 3D dengan pelbagai kepekatan SCAE
menunjukkan morfologi
poligonal mengekalkan
ciri-ciri kondrosit. Penambahan SCAE meningkatkan
percambahan kondrosit
dan keupayaan sel
untuk mengekspresi
gen pengekod kolagen jenis I menurun, sedangkan keupayaan untuk mengekspresikan gen kolagen jenis II, protein teras agrekan dan
sox9 meningkat berbanding
dengan kawalan. Matriks ekstrasel diwarnakan positif oleh toluidin biru,
setara dengan
pengumpulan sGAG
yang lebih tinggi
dalam media kultur dengan penambahan SCAE.
Kajian ini
memperlihatkan bahawa SCAE
boleh memberi
manfaat dalam
pembentukan kultur kondrosit 3D secarain vitro
untuk digunakan dalam terapi kejuruteraan
tisu rawan.
Kata kunci: Kolagen jenis I; kondrosit; kultur tiga dimensi;
osteoarthritis; Stichopus chloronotus
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*Pengarang untuk surat-menyurat; email: mohdheikalyunus@yahoo.com
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